One of the most poorly understood areas of cell biology involves the role of receptor-mediated and bulk-phase endocytosis of cholesterol, coupled to ApoE or ApoB100 containing lipoproteins, in plasma membrane sterol turnover and cellular function. Such cholesterol is processed through the late endosomal/lysosomal compartment of every cell where at least three proteins (lysosomal acid lipase, LAL; Niemann-Pick type C1, NPC1; and Niemann-Pick type C2, NPC2,) act in tandem to metabolize and move this sterol across the limiting membrane of the lysosome into the metabolically active, cytosolic pool. Only when the cholesterol reaches this pool can it become part of the normal flow of sterol through the cell and plasma membrane. Mutations that inactivate any one of these proteins lead to serious abnormalities in cellular cholesterol metabolism that, clinically, may give rise to severe liver and lung disease as well as progressive neurodegeneration. Studies are designed to quantitate the specific abnormalities in cholesterol metabolism that occur with each of these mutations, and to elucidate how these abnormalities in sterol flux lead to cell death in the brain, liver, lung and other organs. These studies use a variety of genetically modified animal models, including lal-/-, npc1-/- and npc2-/- mice, and a spectrum of quantitative techniques to measure, in vivo, the major changes in cholesterol flux that occur in virtually every organ. These measurements include rates of sterol synthesis, lipoprotein-cholesterol uptake and sterol degradation. Changes in the mRNA expression of the target genes of the regulatory proteins LXR and SREBP2 also will be evaluated. In addition, histological techniques, coupled with measurements of the mRNA expression of proteins reflecting macrophage invasion and activation will be used to follow the inflammation and cell destruction that typically occur with these mutations. These molecular and physiological changes in individual tissues, in turn, will be correlated with clinical measurements of pulmonary and hepatic dysfunction, and with the progressive loss of neurons. In addition, several newly described agents that acutely reverse the metabolic effects of one or more of these genetic mutations will be studied utilizing the same quantitative techniques. The ability of these agents to normalize all aspects of cholesterol metabolism in these organs, to prevent the inflammation and cell destruction, and to markedly prolong the life of the mutant mice will be explored in detail. Together, these studies should provide important new information on the pathogenesis of diseases such as Niemann-Pick type C and Wolman disease, which result from disordered cholesterol metabolism, and may even lead to a new form of therapy that can effectively treat such illnesses. PUBLIC HEALTH RELEVANCE: These studies explore in detail a group of disorders where genetic abnormalities inactivate critical proteins involved in the metabolism of cholesterol within cells throughout the body. Utilizing a variety of mutant mouse models and different quantitative techniques, the cellular defects present in these disorders are examined in detail, and, in addition, agents are identified and studied that may correct these defects in cholesterol metabolism and prevent disease. These studies will provide much new information on the pathophysiology of disorders with abnormal intracellular cholesterol metabolism, and may even give rise to effective therapies for disorders such as Niemann-Pick Type C and Wolman disease.